title: Detection and quantitation of two cucurbit criniviruses in mixed infection by real-time RT-PCR.
abstract: Cucurbit chlorotic yellows virus (CCYV) and Cucurbit yellow stunting disorder virus (CYSDV) are whitefly-transmitted criniviruses infecting cucurbit crops inducing similar symptoms. Single and multiplex RT-PCR protocols were developed and evaluated on cucurbit samples collected from commercial greenhouses. Primers and probes were designed from the highly conserved heat shock protein 70 homolog (Hsp70h) gene. Conventional RT-PCR and multiplex RT-PCR assays showed high specificity and suitability for routine screening. TaqMan-based quantitative real-time RT-PCR (RT-qPCR) protocols were also developed for the detection and quantitation of both viruses occurring in single or mixed infection. The assays proved to be highly specific with no cross amplification. RT-qPCR assays showed a 100-1000 times improved sensitivity over conventional RT-PCR. Virus titers in mixed infections were compared to singly infected plants by RT-qPCR. CYSDV and CCYV titers decreased in double infected plants. This paper reports highly specific conventional RT-PCR and quantitative real-time PCR assays for detection, quantitation and differentiation between two closely related cucurbit-infecting criniviruses.
authors.affiliation: Department of Agricultural Sciences, Faculty of Agricultural and Food Sciences, American University of Beirut, Beirut 1107 2020, Lebanon. Electronic address: peterabrahamian@gmail.com.
authors.forename: Peter E
authors.initials: PE
authors.lastname: Abrahamian
authors.forename: Rewa
authors.initials: R
authors.lastname: Seblani
authors.forename: Hana
authors.initials: H
authors.lastname: Sobh
authors.forename: Yusuf
authors.initials: Y
authors.lastname: Abou-Jawdah
mesh_terms: D060085:Coinfection / Q000821:virology*; D029383:Crinivirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D028464:Cucurbita / Q000821:virology*; D017931:DNA Primers / Q000235:genetics; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D015345:Oligonucleotide Probes / Q000235:genetics; D010935:Plant Diseases / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; D018840:HSP70 Heat-Shock Proteins; D015345:Oligonucleotide Probes; D014764:Viral Proteins
keywords: CCYV; CYSDV; Cucurbit chlorotic yellows virus; Cucurbit yellow stunting disorder virus; Multiplex detection; RT-qPCR